This invention relates to use of sFRP fragments and variants thereof to bind to members of the Wnt family of proteins and regulate Wnt biological activity.
Wnt proteins comprise a large family of structurally related, extracellular agents that have a variety of important functions during embryonic development (Cadigan and Nusse, Genes Dev. 11:3286-3305, 1997 and Dale, T. C., Biochem J. 329:209-223, 1998). They specify cell polarity and fate, stimulate proliferation, and contribute to the patterning of tissue in many animal models. Wnt signaling also has been strongly implicated in the development of neoplasia.
A set of secreted Fz-related proteins (sFRP or FRP) recently have been described (Leyns et al., Cell 88:747-756, 1997; Wang et al., Cell 88:757-766, 1997; Rattner et al., Proc. Natl. Acad. Sci. U.S.A. 94:2859-2863, 1997; Finch et al., Proc. Natl. Acad. Sci. U.S.A. 94:6770-6775, 1997; Salic et al., Development 124:4739-4748, 1997; Melkonyan et al., Proc. Natl. Acad. Sci. U.S.A. 94:13636-13641, 1997; Pfeffer et al., Int. J. Dev. Biol. 41:449-458, 1997; Mayr et al., Mech. Dev. 63:109-125, 1997; Wolf et al., FEBS Lett. 417:385-389, 1997; Xu et al., Development 125:4767-4776, 1998; Chang et al., Hum. Mol. Genet. 8:575-583 1999; and Abu-Jawdeh et al., Lab. Invest. 79:439-447, 1999). These proteins consist of approximately 300 amino acids, including a CRD (cysteine rich domain) that is typically 30-50% identical to the CRDs of Fz family members. The carboxyl-terminal portion of these proteins often contains segments rich in positively charged residues, and two (sFRP-1 and FrzB/sFRP-3) were reported to bind tightly to heparin (Finch et al., Proc. Natl. Acad. Sci. U.S.A. 94:6770-6775, 1997 and Hoang et al., J. Biol Chem. 271:26131-26137, 1996). The CRD has been also found to be the Wnt binding site based on several experiments in which the Fz CRD conferred Wnt binding and/or responsiveness (Hsieh et al., Proc. Natl. Acad. Sci. U.S.A. 96:3546-3551, 1999; Bhanot et al., Nature 382:225-230, 1996; and He et al., Science 275:1652-1654, 1997).
The invention stems from the discovery that sFRP and fragments thereof can bind to members of the Wnt family of proteins, and furthermore that these molecules have a biphasic effect on Wnt activity. At high concentrations these proteins inhibit Wnt activity and at low concentrations these proteins increase in Wnt biological activity. Furthermore, fragments of sFRP that do not contain the CRD domain are provided and these fragments are shown to bind to Wnt proteins and modulate Wnt biological activity.
Accordingly, the invention provides fragments of sFRP which are able to bind to Wnt thereby modulating Wnt biological activity. These sFRP fragments may (SEQ ID NOS: 5-7) or may not (SEQ ID NO: 8) contain the CRD of sFRP. Because these fragments bind to Wnt these fragments, and variants thereof, can be used to screen for other molecules that bind to Wnt and modulate Wnt activity.
The invention also provides methods of using sFRP-1 and fragments thereof to increase Wnt biological activity. The increase in Wnt activity is desirable for treating developmental disorders that are associated with decreased Wnt biological activity as well as for inducing the development of neoplasias which is desirable in experimental models for the study of tumor growth.
The invention also provides methods of using sFRP without the CRD domain to increase or decrease Wnt biological activity depending upon the amount provided. Such methods are useful for treating disorders associated with increased Wnt biological activity and for the suppression of tumor growth. Furthermore, the finding that sFRP fragments without the CRD domain bind to Wnt proteins allows for the development of screening assays which identify small molecules or other compounds which may block sFRP/Wnt binding or enhance sFRP/Wnt binding. Thus, for example, the invention provides methods of identifying small molecules or binding proteins that bind either Wnt or fragments of sFRP without the CRD and disrupt sFRP/Wnt binding.
Accordingly, another aspect of the invention provides methods of modulating Wnt protein biological activity. These methods involve contacting at least one Wnt protein with least one sFRP fragment or variant thereof and producing an increase Wnt biological activity.
Yet another aspect of the invention provides a sFRP fragment that does not contain the CRD portion of sFRP (SEQ ID NO: 8), but yet maintains Wnt binding activity. Accordingly this fragment and variants thereof can be used to screen for other molecules that bind to Wnt and modulate Wnt biological activity.
When used to modulate Wnt biological activity the fragments described above can be used to further characterize the biological role that Wnt plays in the various developmental processes. Furthermore, these fragments can also be used to modulate conditions associated with abnormal Wnt biological activity.